In-cell architecture of an actively transcribing-translating expressome

Structural biology performed inside cells can capture molecular machines in action within their native context. Here we developed an integrative in-cell structural approach using thegenome-reduced human pathogenMycoplasma pneumoniae. We combined whole-cellcrosslinking mass spectrometry, cellular cryo-electron tomography, and integrative modelingto determine an in-cell architecture of a transcribing and translating expressome at sub-nanometer resolution. The expressome comprises RNA polymerase (RNAP), the ribosome,and the transcription elongation factors NusG and NusA. We pinpointed NusA at theinterface between a NusG-bound elongating RNAP and the ribosome, and propose it canmediate transcription-translation coupling. Translation inhibition dissociatedthe expressome,whereas transcription inhibition stalled and rearranged it. Thus, the active expressomearchitecture requires both translation and transcription elongation within the cell.